The central question of this proposal is: How is cell polarity regulated by Wnt5a and how do these mechanisms impact cancer metastasis? This proposal will build on recent findings revealing regulation of cell adhesion molecule depalmitoylation by Wnt5a signal activation promotes cancer cell invasion. Palmitoylation is the addition of the 16 carbon fatty acid palmitate to proteins on cysteine residues. Wnt5a treatment of melanoma cells induces depalmitoylation of the cell adhesion molecules MCAM in melanoma cells. MCAM mutations that block palmitoylation cause polarized localization of MCAM in the absence of exogenous Wnt5a as well as increased invasion of cells in 3D collagen and in xenograft tumor assays. Wnt5a signaling decreases palmitoylation through a mechanism requiring the acyl protein thioesterase APT1 and inhibition of APT1 using shRNA or pharmacological inhibitors blocks cell migration and invasion. The proposed studies will begin to explore how cancer metastasis is regulated by Wnt5a signaling and elucidating the molecular mechanism of protein palmitoylation regulated by Wnt5a during polarized cell migration. In the first aim the hypothesis that a known downstream component of the noncanoncial Wnt pathway interfaces with the core palmitoylation machinery to regulate depalmitoylation. The mechanism by which Wnt5a induces protein depalmitoylation will be investigated taking both a candidate approach to identify the Wnt signaling components required for Wnt5a induced protein depalmitoylation. The second aim will determine the role of the protein palmitoylation cycle during cell migration and invasion in vitro and in vivo. Mutations in palmitoyl transferases identified in human tumor samples have impaired ability to palmitoylation MCAM and to suppress cell invasion in collagen. The impact of these mutations in vivo will be determined by xenograft tumor assays. Cell behavior will be studied using in vitro migration and invasion assays and protein localization and directional migration will be analyzed by time lapse imaging. Other palmitoylated proteins that are regulated by Wnt5a will be biochemically purified and identified using mass spectrometry providing a broad view of the cellular pathways regulated by Wnt5a induced depalmitoylation. The proteins identified in the pathway are being examined in patient tumor samples.